Megaphages harbor mini-Cas proteins ideal for gene editing

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A megaphage (left), a member of a bacteriophage family Biggiephage, injects its DNA — including genes for CasΦ (red) — into bacterial cells to turn the bacteria against the phage’s competitor (top). The reddish Pac-Man-like figures are CasΦ proteins, enzymes that cut up viral DNA. The genome of the bacterium is shown in purple. (UC Berkeley image by Basem Al-Shayeb and Patrick Pausch)

The new Cas proteins were found in the largest known bacteria infecting viruses, called bacteriophages, and are the most compact working Cas variants yet discovered — half the size of today’s workhorse, Cas9. 

CRISPR is a natural process that’s long functioned as a bacterial immune system. Originally found defending single-celled bacteria and archaea against invading viruses, naturally occurring CRISPR uses two main components. The first is CRISPR, short for ”clustered regularly interspaced short palindromic repeats”. The second is Cas, or CRISPR associated proteins, which chop up DNA like molecular scissors.

As one of the smallest Cas proteins known to date, the newly discovered CasΦ (Cas-phi) has advantages over current genome-editing tools when they must be delivered into cells to manipulate crop genes or cure human disease. 

Cas Proteins 

The CasΦ protein was first discovered last year by Al-Shayeb in the laboratory of Jill Banfield, a UC Berkeley professor of earth and planetary science and environmental science, policy, and management. The megaphages containing CasΦ were part of a group they dubbed Biggiephage and were found in a variety of environments, from vernal pools and water-saturated forest floors to cow manure lagoons.

The ability of CasΦ to cut double-stranded DNA is a big plus. All other compact Cas proteins preferentially cut single-stranded DNA. So, while they may fit neatly into compact delivery systems like AAV, they are much less useful when editing DNA, which is double-stranded, inside cells.

As was the case after Cas9’s gene-editing prowess was first recognized in 2012, there is a lot of room for optimizing CasΦ for gene editing and discovering the best rules for designing guide RNAs to target specific genes, Pausch said.

Other co-authors of the paper are Ezra Bisom-Rapp, Connor Tsuchida, Brady Cress, and Gavin Knott of UC Berkeley and Zheng Li and Steven E. Jacobsen of UCLA. The researchers were funded, in part, by the Paul G. Allen Frontiers Group, National Institutes of Health Somatic Cell Genome Editing Consortium, and National Science Foundation.

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Materials provided by the University of California – Berkeley

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